Isolation of Nuclei and Protein Extraction

The nuclei were prepared from chickpea seedlings as described [20]. About 20 g of the tissue was ground into powder in liquid nitrogen with 0.3% (w/w) PVPP and immediately transferred into an ice-cold 500-mL beaker containing 200 mL of ice-cold 1X HB (10 mM Trizma base, 80 mM KCl, 10 mM EDTA, 1 mM spermidine, 1 mM spermine, 0.5 M sucrose, pH 9.5) plus 0.15% 2-ME and 0.5% Triton X-l00. The contents were gently stirred for 30 min for complete lysis of organeller membranes. This suspension was filtered through four layers of cheesecloth and two layers of Mira cloth into an ice-cold 250-mL centrifuge bottle. The homogenate was pelleted by centrifugation with a fixed-angle rotor at 1800g at 4°C for 20 min. The supernatant fluid was discarded and the pellet was gently resuspended in 30 mL of ice-cold wash buffer (1X HB minus Triton X-100). To remove the particulate matter remaining in the suspension, the resuspended nuclei were filtered into a 50-mL centrifuge tube through two layers of Mira cloth by gravity. The content was centrifuged at 57g, 4°C for 2 min to remove intact cells and tissue residues. The supernatant fluid was transferred into a fresh centrifuge tube and the nuclei were pelleted by centrifugation at 1800g, 4°C for 15 min in a swinging bucket centrifuge. The pellet was washed two additional times by resuspension in wash buffer followed by centrifugation at 1800g, 4°C for 15 min. Nuclear proteins were prepared from the nuclei-enriched pellet using TriPure Reagent (Roche) according to the manufacturer's instructions. The final protein pellet was resuspended in IEF sample buffer [8 M urea, 2 M thiourea, 2% (w/v) CHAPS]. The protein concentration was determined using the 2-D Quant kit (Amersham Biosciences).

How To Win Your War Against Allergies

How To Win Your War Against Allergies

Not Able To Lead A Happy Life Because Of Excessive Allergies? Want To Badly Get Rid Of Your Allergy Problems, But Are Super Confused And Not Sure Where To Even Start? Don't Worry, Help Is Just Around The Corner Revealed The All-In-One Power Packed Manual Containing Ample Strategies And Little-Known Tips To Get Rid Of Any Allergy Problems That Are Ruining Your Life Learn How You Can Eliminate Allergies Completely Reclaim Your Life Once Again

Get My Free Ebook


Post a comment